Recent developments in the field of high-throughput screening technologies have broadened the range of applications, and a large choice of materials, formats, immobilization methods, and detection systems is now available for both in solution- and cell-based assays (Sundberg, 2000).
Novel analytical and screening technologies make often use of a solid support (in the form of plates, tubes, or beads) onto which a reactant is immobilized and then exposed to one or more reactions, generating a measurable signal on the support itself or in the reaction solution. The immobilization step gives the possibility to simplify the separation amongst different reactions and/or reagents, and may also allow the amplification of the signal. Such an effect can be obtained, for example, by impregnating the solid support with scintillant molecules that, excited by the radiation emitted from the immobilized molecules, multiply or alter consistently the signal generated by a labeling group alone (Meza, 2000).
Nowadays, a variety of inorganic or organic polymer core compositions enables the efficient immobilization of many ligands, as well as the scintillant impregnation, facilitating the separation and/or the sensitive detection, such as scintillation and wavelength-specific fluorescence or absorbance.
Amongst the solid phase assay based on these properties, the Scintillation Proximity Assay (SPA) has been widely applied to many biological assays involving the use of radiolabeled molecules like enzyme substrates, antibodies, proteins, or DNA. The technology relies on the observation that β rays emitted from molecules labeled with weak radioisotopes (such as 3H, 125I, 33P, 35S) travel only a limited distance in an aqueous environment before the energy is dissipated. These emissions can be detected with great sensitivity if the radioisotopically labeled molecules are brought into close proximity to solid support containing a scintillant compound (such as 2,5-diphenyloxazole), thereby causing a specific emission. Radiolabeled molecules remaining free into solution are undetected because they are too distant from the scintillating solid phase. Such support onto which the ligand is immobilized is usually a polymer (polystyrene, polyvinyltoluene, or yttrium-derived polymers), and can be in the form of microspheres (EP154734, U.S. Pat. No. 4,271,139) or 96-/384-wells microplates (EP576090).
SPA beads coated with many biologically relevant ligands are commercially available (Amersham). For example, SPA beads can enable the sensitive detection of radiolabeled antibodies using immobilized Protein A, of radiolabeled GST (Glutathione-S-Transferase) fusion proteins using immobilized glutathione, or of biotinylated radiophosphorylated kinases using immobilized streptavidin. Another SPA-based commercially available technology (FlashPlate®, NEN Life Science Products) makes use of polystyrene microplates wherein wells are covered with a layer of polystyrene-based scintillant. It is however not known from prior art that the immobilization of aminoglycosides can be performed to establish SPA-based assays.
Aminoglycosides are hydrophilic multiply charged compounds, closely related to carbohydrates, whose backbone structure consists of an aminocyclitol ring saturated with amine and hydroxyl substitutions in specific positions. Aminoglycosides show high flexibility, high solublility in water, and relative insolublity in lipids, due to the basic, strongly polar groups they contain (Zembower et al., 1998).
The aminoglycosides can be divided into structural types based on the position of their glycosidic linkages, as well as on the presence and the substitutions of the 2-deoxystreptamine group. Certain compounds, often considered as aminoglycosides, actually do not contain aminosugars, thus the term aminocyclitol has been introduced to describe this entire group of molecules rather than the less precise “aminoglycoside” term, which is commonly accepted and used henceforth for simplicity.
Aminoglycosides like neomycin, dibekacin, gentamicin, tobramycin, kanamycin, amikacin, and streptomycin are well known for their antibiotic properties. These molecules are able to bind in vivo and in vitro a variety of biological ligands.
At the level of cellular membrane, aminoglycosides bind polyphosphoinositides, negatively charged phospholipids, which constitute a minor component of membrane bilayers, perturbing the permeability and other biochemical properties of the cell wall (Mingeot-Leclercq et al., 1992).
At the intracellular level, a growing number of nucleic acid structures, RNAs in particular, has been shown to be bound and modulated in their action by aminoglycosides. Examples of RNA targets recognized and altered by aminoglycosides are ribosomal RNAs, RNA aptamers, ribozymes, and other non-coding RNA sequences (Walter et al., 1999; Schroeder et al., 2000). Aminoglycosides can be modified by bacterial enzymes, providing resistance features to organisms that harbor these enzymes, due to the diminished antibacterial ability of the altered molecules (Llano-Sotelo et al., 2002).
The specificity of the aminoglycoside-phosphoinositide interaction has been studied at the structural level, demonstrating the involvement of both hydrophobic and hydrophilic interactions which determine also a toxic effect for the inner ear and kidney cells into patients (Schacht, 1986).
Neomycin, like other antibiotics of the same family, is known from late '70 to be efficiently bound by phosphoinositides. Such interaction has been exploited to extract and purify polyphosphoinositides from crude extracts by affinity chromatography on a support where neomycin was immobilized (Schacht, 1978). The immobilization of aminoglycosides has been achieved on solid phases like sepharose (WO9008584, JP61976418), polystyrene microtiter plates (Sachetelli et al., 1998), or medical devices (EP372130). Other applications based on the same principle are the selective separation and aggregation of liposomes containing polyphosphoinositides (Riaz et al., 1989; Van Bambeke et al., 1995), or the detection of polyphosphoinositides into cell membrane and intracellular vescicles (Arbuzova et al., 2000).
Moreover, fluorescently labeled aminoglycosides are known to be useful in screening RNA-binding compounds (WO9635811). Compositions facilitating the uptake of aminoglycoside antibiotic, and containing phosphoinositide polyphosphate or derivative and a labeled polyamine, like aminoglycosides, are known in the prior art (WO0018949). These compositions have been described as helping the visualization of the uptake and localization of aminoglycosides, the screening for compounds that minimize the cytotoxicity of aminoglycoside antibiotics to mammalian cells, for monitoring, calcium flux in a cell, and for screening of agonists and antagonists for proteins, in particular kinases, interacting with phosphoinositides. Either the aminoglycosides or phosphoinositides can be covalently bound to a fluorescent compound.
The binding properties of aminoglycosides allow the clearance of bacteria by inhibiting protein synthesis, decreasing the fidelity of messenger RNA translation, and disrupting the integrity of the bacterial cell membrane. The interaction of aminoglycosides with biological ligands, thus, has considerable physiological effects, and methods for identifying and/or quantifying Aminoglycoside Binding Molecules (ABMs), enzymes modifying such molecules, and compounds modulating the interaction between ABMs and either enzymes or aminoglycosides have many important applications, in particular if compatible with high throughput formats.
An example of physiological mechanism which can be studied by exploiting the interaction between aminoglycosides and biological ligands is constituted by the phosphorylation of cell membrane phosphoinositides.
Phosphoinositides have a basic structure, termed phosphatidylinositol, consisting of diacyl-glycerol linked by phosphodiester bond to the 1′ position of an inositol head group. The acyl chains of diacylglycerol (typically stearyl-arachidodyl) are inserted into the inner leaflet of the membrane bilayer. The inositol head group, which is cytosolic, may be further phosphorylated on the 3′, 4′, 5′, or any combination of these, by enzymes called phosphoinositide kinases (PIKs) which are specific for a single position on the inositol ring. Such different phosphorylation states characterize molecules having highly specific properties. In particular, the phosphoinositide 3-kinases (PI3Ks) form a family of ubiquitously expressed enzymes that, through phosphorylating membrane inositol lipids in the 3′ position of the inositol ringl and the consequent generation of phospholipid second messengers, play a key role in the regulation of many cellular processes (such as motility, proliferation, differentiation, apoptosis, membrane transport, and carbohydrate metabolism), representing one of the major pathways of intracellular signal transduction (U.S. Pat. No. 6,017,763; Leevers et al., 1999; Stein and Waterfield, 2000; Hinchliffe, 2001; Comer and Parent, 2002; Simonsen et al., 2001).
To date, eight mammalian PI3Ks have been identified, divided into three main classes (I, II, and III) on the basis of sequence homology, structure, binding partners, mode of activation, and substrate preference in vitro. All PI3Ks share a kinase domain located towards the C-terminus of the enzyme, a scaffold-like helical region, and a C2 domain known to bind phospholipids. The N-terminus of the enzyme, where interactions with adapter subunits and other proteins are believed to occur, is highly variable. Many other enzymes have been identified whose catalytic domains closely resemble those of PI3Ks, like the phosphoinositide 4-kinases (PI4Ks), DNA-dependent protein kinases, and the mTOR kinase.
Even though other phosphatidylinositols having a 3′ group available for the phosphorylation can be modified by PI3Ks, the major cellular substrate is phosphatidylinositol (4,5)-bisphosphate (also indicated as PtdIns(4,5)P2, PIP2 or PI(4,5)P2). Upon activation of PI3K by several G Protein Coupled Receptors (GPCR) agonists, growth factors or inflammatory cytokines, PI(4,5)P2 is converted into phosphatidylinositol (3,4,5)-trisphosphate (also indicated as PI(3,4,5)P3, PtdIns(3,4,5)P3, or PIP3). This latter molecule has been characterized in vivo as a second messenger responsible for a wide variety of signaling events including phosphorylation and activation of several downstream effectors of PI3K. Moreover, several proteins, containing the Pleckstrin homology (PH) or the Phox homology (PX) domains and requiring membrane association for their function, interact directly with the cell membrane by binding to phosphoinositides with a broad range of specificity and affinity for different phosphorylation state (Xu et al., 2001).
Four class I PI3Ks (α,β,γ, and δ isoforms) have been described in humans and other mammals in connection with distinct or overlapping cellular functions, and are the PI3Ks better characterized at both structural and functional level. PI3Kα, PI3Kβ and PI3Kδ are widely expressed and activated by interacting, through SH2 domain-containing adapter molecules, with growth factor receptor tyrosine kinases and other intracellular phosphotyrosine-containing proteins. In contrast, PI3Kγ is only expressed in hematopoietic cells (especially in leukocytes), and it is the only isoform that has been shown to respond, through an adapter molecule not containing the SH2 domain, to G-protein coupled receptors through the interaction and activation with G-protein βγ-subunits. Interestingly, mice lacking PI3Kγ show, amongst other phenotypes, a clear defect in leukocyte migration and are less susceptible of septic shock, suggesting a role of PI3Kγ on cell migration (Hirsch et al., 2000). The functional specialization of PI3K isoforms suggests that isoform-selective inhibition with acceptable side effects might be possible and therapeutically useful in many conditions. PI3Kγ appears as an attractive drug target candidate for the treatment of inflammatory processes involving leukocyte migration, as malignant diseases and other diseases with an inflammatory or immune component.
Two molecules have been mostly studied as PI3Ks inhibitors: Wortmannin, previously known as an inhibitor of respiratory-burst activity (Arcaro and Wymann, 1993) and LY294002 (Powis et al., 1994). Although Wortmannin and LY294002 have been widely used to elucidate the biological functions of PI3Ks activation at a cellular level in short term assays, these compounds have specific properties limiting their pharmaceutical potential. Both Wortmannin and LY294002 show a poor selectivity, since different class I kinases are inhibited at comparable concentrations (Stein and Waterfield, 2000). Moreover, Wortmannin, binds PI3Kγ ATP-binding site irreversibly at nanoMolar concentrations but it is a molecule quite unstable, while LY294002 binds PI3Kγ ATP-binding site competitively at microMolar concentrations but it is a molecule with solubility problems. A number of chemical modifications of Wortmannin and LY294002 have been made, only a few of them providing molecules significantly more potent than the parent compound (Creemer et al., 1996).
Various technologies have been tested for improving the throughput of screening for PI3Kγ, or other PI3K related kinases, and identify more potent and specific inhibitors. All these methods have in common the incubation of the kinase with a substrate (usually phosphoinositide), a radiolabeled precursor ([γ-32P] ATP or [γ-33P] ATP) and the potential inhibitor, but the way these basic components are arranged or modified to establish the assay can have a profound effect on the throughput and the sensibility of the assay.
The prior art discloses different systems for identifying compound interfering with the phosphorylation activity of PI3K-related kinases, by means of antibodies specific for a moiety conjugated to the potential inhibitor (WO9855602), the analysis of alterations in motility of cells exposed to the potential inhibitor (WO9935283), lipid extraction combined to chromatographic separation (Ward, 2000), directly labeled aminoglycosides (WO0018949), or thin layer chromatography (Frew et al., 1994). Such assays are laborious to carry out, difficult to automate and generate problems and expenses due to the radioactive waste.
Some high throughtput screening technologies have been developed for studying enzymes modifying phosphoinositides. FlashPlate® microplates covered with [3H] PI(4,5)P2 have been generated by incorporating this molecule through covalent attachment and/or by hydrophobic interaction and used to test Phospholipase C, an enzyme that catalyzes the hydrolysis of phosphoglycerides into diacylglycerols and phosphorylated alcohols. The assay, however, measures the reduction of radioactivity in the microplate due to the generation of [3H]-inositol which migrates in the acqueous phase and then is removed (WO9932655). Alternatively, phosphoinositide have been bound covalently on the scintillant substrate using a linker moiety, like polyethylene glycol or succinimide, providing composition useful for assaying phosphatidylinositol kinases and for screening compounds inhibiting these enzymes. (WO0000584).
However there is no indication that aminoglycoside covered scintillating supports can be used as a general tool for identifying and/or quantifying Aminoglycoside Binding Molecules (ABMs), enzymes modifying such molecules, compounds modulating the interaction between ABMs and either enzymes or aminoglycosides.